Serotonin acetyltransferase (arylalkylamine-A/-acetyltransferase, AANAT) governs the diurnal rise and fall of melatonin. In humans the rhythmic synthesis of melatonin is controlled by the phosphorylation state of AANAT. At night, AANAT is phosphorylated at two sites (N- and C- terminal) and during the day AANAT is mainly present in its unmodified form. Phosphorylation of AANAT promotes binding to 14-3-3 gamma which increases enzyme stability (e.g. resistance to proteases) and activity thus promoting melatonin synthesis. This proposal focuses on the use of the nonhydrolyzable pSer/pThr mimetic, phosphono-difluoro-methylene-L-alanine (Pfa), and protein semi-synthesis to further explore the cellular regulation of AANAT. First, the effectiveness of Pfa to serve as a phospho-mimetic will be examined by in vitro binding and kinetic assays. Next, semi- synthetically prepared Pfa modified AANAT will be used as bait to identify novel interacting proteins (e.g. phosphatases). Finally, semi-synthetic fluorescence resonance energy transfer (FRET) labeled AANAT will be prepared and used for cellular microinjection assays aimed at measuring real time enzyme stability and identifying small molecules and enzymes that effect enzyme stability. This semi-synthetic approach will lead to a better understanding of AANAT regulation and circadian rhythms. [unreadable] [unreadable] [unreadable]